The objective of the proposed research program is to study the role of the cysteine residues in the different facets of hemoglobin structure and function (tetramer formation, oxygen affinity, cooperative oxygen binding, etc.). While not invariant throughout the phylogenetic scale, the cysteine residues have not been found substituted for in abnormal human adult hemoglobins. There are only six cysteine residues in each adult hemoglobin molecule and they are located at important sites in the tetramer (at a contact point between subunits and next to a distal histidine). Therefore, chemical modification of these amino acids will be used to determine their role in hemoglobin structure and function. Hemoglobins A, F, and S have been chosen because of the differences which exist in the primary structure of the beta-like subunit of these molecules. The different cysteine residues will be modified by reaction with compounds which bind either covalently or noncovalently. After separation of the alpha and beta hemoglobin subunits and modification of the desired cysteine residue(s), the subunits will be recombined. Products from these mixtures which resemble normal hemoglobin A chromatographically and electrophoretically will be isolated and their equilibrium properties during oxygen binding will be compared with respect to differences in primary sequence of the beta-like subunit. The cysteine residues will also be used as sites for spin-label probes which will be used to monitor structural changes which take place within the different hemoglobin molecules during reversible oxygen binding. In addition, these spin-labeled derivatives will be used to detect structural changes on the interior of the hemoglobin molecule following binding of the effector of oxygen affinity, 2,3-diphosphoglyceric acid. Also, the spin-labeled derivative of hemoglobin will be used to monitor changes which occur on the interior of the molecule upon aggregation of the deoxy form. The information obtained from these studies should help elucidate the nature of the structural differences between these three hemoglobins and therefore provide further information concerning their functional differences, such as the involvement of hemoglobin S in sickle cell anemia.